Project Summary Herpes simplex virus 1 (HSV-1) and HSV-2 cause millions of chronic infections. These viruses cause epithelial oral ?cold? sores and genital lesions, which recur lifelong due to reactivation from a latent viral reservoir in neurons. The epidemiology of genital herpes has undergone a significant transformation over the past two decades, with the emergence of HSV-1 as a leading cause of first-episode genital herpes. The rise of genital HSV-1 coincides with a decline in seroprevalence of HSV-1, particularly among youth, and with changing sexual behaviors (13, 23). This shift raises the possibility that HSV-1 may face new selective pressures and undergo genetic adaptation as it moves from the oral to the genital niche. We propose to conduct a genome- wide analysis of HSV-1 variation in human infections, to observe how the virus adapts to the human genital niche during the first year of infection, and to discern how the virus is affected by the laboratory culture that is required for further studies in vitro, or in vivo in animal models. These data will reveal the degree to which HSV-1 undergoes bottlenecks or develops new variations in humans or in culture. The experiments in this exploratory R21 proposal provide the foundation for a future R01 proposal aimed at investigating how HSV-1 genetic variations impact clinical disease outcomes in patients. Insights on viral genomic variation will be obtained using Illumina sequencing of longitudinal HSV-1 genital and oral shedding swabs, which are being collected as part of an ongoing clinical study, P01AI030731. In Aim 1, we will use sensitive enrichment and viral genome sequencing techniques to assess HSV-1 genome diversity directly from human shedding. We will compare HSV-1 genomes from genital and oral swabs, collected at primary infection and during any recurrences over the course of a year. These will provide insights on viral genetic diversity between individuals, as well as within-host diversity over the first year of infection. Since P01AI030731 includes sub-studies on mucosal and humoral immune responses during the first year of infection, this proposal will yield additional data on how viral antigen variation correlates with the epitopes detected in each person's immune repertoire. In Aim 2, we will address whether HSV-1 strains that are cultured in vitro accurately reflect the viral genomic diversity shed by infected humans. We will do this by comparing viral genomes derived from cultured stocks to the genomes derived from the matched swab samples in Aim 1. This will establish the foundation for future studies that utilize cultured HSV-1 strains for in vitro and in vivo animal studies of viral pathogenesis, antiviral drug discovery, and vaccine development.